Anhydro sugar derivatives of indolocarbazoles

ABSTRACT

The present invention concerns novel sugar derivatives of indolocarbazoles and pharmaceutical formulations thereof which exhibit topoisomerase-1 activity and are useful in inhibiting the proliferation of tumor cells.

RELATED APPLICATIONS

This application claims priority benefit under Title 35 §119(e) of U.S.provisional Application No. 60/238,696, filed Oct. 6, 2000. The contentsof which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention describes sugar derivatives of indolocarbazoleswhich exhibit topoisomerase-I activity and are useful in inhibiting theproliferation of tumor cells.

BACKGROUND

Topoisomerases are vital nuclear enzymes which function to resolvetopological dilemmas in DNA, such as overwinding, underwinding andcatenation, which normally arise during replication, transcription andperhaps other DNA processes. These enzymes allow DNA to relax by formingenzyme-bridged strand breaks that act as transient gates or pivotalpoints for the passage of other DNA strands. Topoisomerase-targetingdrugs appear to interfere with this breakage-reunion reaction of DNAtopoisomerases. In the presence of topoisomerase active agents, anaborted reaction intermediate termed a ‘cleavable complex’ accumulatesand results in replication/transcription arrest, which ultimately leadsto cell death. The development of topoisomerase I active agentstherefore offers a new approach to the multi-regimental arsenal oftherapies currently used in the clinic for the treatment of cancer.

An article in Cancer Chemother. Pharmacol [1994, 34 (suppl): S 41-S 45]discusses topoisomerase I active compounds that have been found to beeffective clinical anti-tumor agents. Structurally these clinicalcandidates are related to the alkaloid camptothecin.

Indolo[2,3-a]carbazole alkaloids such as rebeccamycin (U.S. Pat. Nos.4,487,925 and 4,552,842) and its water-soluble, clinically-activeanalog, 6-(2-diethylaminoethyl)rebeccamycin (U.S. Pat. No. 4,785,085),are useful antitumor agents which target DNA. Furthermore,fluoroindolocarbazoles have been disclosed in WO 98/07433 to act asantineoplastic agents with topoisomerase I inhibitory activity.

Indolo[2,3-a]carbazole derivatives related to the Rebeccarnycin classare disclosed (EP Appl. 0 545 195 B1 and 0,602,597 A2; Cancer Research1993, 53, 490-494; ibid 1995, 55, 1310-1315) and claimed to exhibitanti-tumor activity. However, the major mechanism of action of thesederivatives may not be like camptothecin, which acts as a topoisomeraseI poison. Other indolocarbazoles related to those mentioned above aredisclosed in WO 95/30682 and are claimed to exhibit anti-tumor activity.

Hudkins, et al. disclosed a series of fused pyrrolocarbazoles (WO96/11933 and U.S. Pat. No. 5,475,110) and showed in vitro biologicaldata such as inhibition of neuronal choline acetyltransferase (CHAT) andprotein kinase C (PKC) inhibition for some compounds. U.S. Pat. No.5,468,849 discloses certain fluororebeccamycin analogs as usefulantitumor agents, along with a process for their production byfluorotryptophan analog feeding of a rebeccamycin-producing strain ofSaccharothrix aerocolonigenes, particularly Saccharothrixaerocolonigenes C38,383-RK2 (ATCC39243). Glicksman, et al. discloseindolocarbazole alkaloids (U.S. Pat. No. 5,468,872) which are differentin structure from those of the present invention. Kojiri, et al.disclose indolopyrrolocarbazoles having a dissacharide substituent (WO96/04293) which are not related to the anhydrosugar indolocarbazoles.Weinreb, et al (Heterocycles 1984, 21, 309) and Kleinschroth, et al(U.S. Pat. No. 5, 043,335) have disclosed indolopyrrolocarbazolederivatives with a bridging furan moiety and McCombie, et al. (Bioorg.Med. Chem. Lett. 1993, 3, 1537) have reported a more functionalizedbridged furan. Similarly, Wood, et al. have reported the total synthesisof (+)-K252a (J. Am. Chem. Soc. 1995, 117, 10413), a related,naturally-occuring indolocarbazole alkaloid which has demonstrated PKCinhibitory activity.

Danishefsky, et al., during the course of their first total synthesis ofstaurosporine (J. Am. Chem. Soc. 1996, 118, 2825), describe thesynthesis of an intermediate N12, N13-bridged indolopyrrolocarbazole.Indolocarbazole derivatives with the nitrogens linked by a three-atombridge have been reported to be potent PKC inhibitors (S.F. Vice, et al.Bioorg. Med. Chem.Lett. 1994, 4, 1333). The synthesis of simpleindolocarbazole derivatives with C1′, C-5′-bridging or C1′, C3′-bridgingglycosides have also been reported in the literature (B. M. Stolz, J. L.Wood Tetrahedron Lett. 1995, 36, 8543, B. B. Shankar, S. W. McCombieTetrahedron Lett. 1994, 35, 3005, respectively). Prudhomme, et al.disclose a series of antitumor indolocarbazoles derived fromrebeccamycin which exhibit a carbohydrate attached to the two indolenitrogens, and reported their cytotoxicity and their topoisomerase I andPKC inhibitory activities to be in the millimolar to micromolar range(Bioorg. Med. Chem. 1998, 6, 1597). There is yet a need for novel andpotent cytotoxic compounds useful for inhibiting topoisomerase Iactivity.

SUMMARY OF THE INVENTION

Thus according to a first embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) and pharmaceuticallyacceptable salts and solvates thereof, useful for inhibitingtopoisomerase I and the proliferation of tumor cells

wherein:

R is hydrogen, OH, OC₁₋₇alkyl, NH₂, N(C₁₋₃alkyl)₂ or C₁₋₇alkyl, whereinsaid C₁₋₇alkyl is optionally substituted with one or more substituentsselected from the group consisting of halogen, CN, OR⁹ and NR⁹R¹⁰;

Q is O, S, CH₂ or NR^(5a);

R⁵ and R^(5a)are each independently selected from the group consistingof

 provided that

if Q is NR^(5a), then either R⁵ or R^(5a) must be hydrogen;

R¹, R², R³ and R⁴ are each independently selected from the groupconsisting of hydrogen, C₁₋₇alkyl, C₃₋₇cycloalkyl, halogen, azido,NR⁹R¹⁰, NHC(O)NR⁹R¹⁰, NHC(O)OR⁹, C(O)OR⁹, SR⁹ and OR⁹, wherein said C₁₋₇alkyl is optionally substituted with one or more substituents selectedfrom the group consisting of halogen, CN, OR⁹, SR⁹ and NR⁹R¹⁰;

or R¹ and R² together form ═N—OH, ═O or —NR⁹R¹⁰;

or R³ and R⁴ together form ═N—OH, ═O or —NR⁹R¹⁰;

W is selected from the group consisting of hydrogen, C₁₋₇alkyl,C₃₋₇cycloalkyl, halogen, azido, NR⁹R¹⁰, NHC(O)NR⁹R¹⁰, NHC(O)OR⁹, N—OH, Oand OR⁹, wherein said C₁₋₇alkyl is optionally substituted with one ormore substituents selected from the group consisting of halogen, CN, OR⁹and NR⁹R¹⁰;

R⁷ and R⁸ are independently OH or H or together form ═O;

R⁹ and R¹⁰ are independently selected from the group consisting ofhydrogen, C₁₋₇alkyl and C₃₋₇cycloalkyl, wherein said C₁₋₇alkyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, CN, OH, O—C₁₋₇alkyl, NH₂ and N(C₁₋₃alkyl)₂;or

R⁹ and R¹⁰ together with the nitrogen atom to which they are attachedform a non-aromatic 5-8 membered heterocycle containing one or two ofthe same or different heteroatoms selected from the group consisting ofO, N and S; and

X¹, X^(1′), X² and X^(2′) are independently selected from the groupconsisting of hydrogen, halogen, cyano, OR⁹, —CF₃, alkylcarbonyl,C-₁₋₇alkyl, nitro, NR⁹R¹⁰, SR⁹ and C(O)OR⁹; wherein said C₁₋₇alkyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, CN, OR⁹, SR⁹ and NR⁹R¹⁰.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R^(5a) is not H.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R^(5a) isformula (C) or (A).

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R⁵ is formula(A).

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R⁵ is formula(B).

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R⁵ is formula(C).

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R⁵ is formula(D).

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein Q is NR^(5a) andR^(5a) is H or wherein Q is S.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R is H, OH orNH₂.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R is H.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R⁷ and R⁸together are ═O.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein X^(2′) and X²are each F and X¹ and X^(1′) are each H.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein X² is F andX^(2′), X¹ and X^(1′) are each H.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein X^(2′) is F andX², X¹ and X^(1′) are each H.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein X^(2′), X², X¹and X^(1′) are each F.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein X^(2′) and X²are each H and X¹ and X^(1′) are each F.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein R¹, R², R³ andR⁴ are independently selected from the group consisting of H, F and OR⁹wherein R⁹ is H.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein W is fluorine.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein the bondattaching R⁵ to N is in the β designation when R⁵ is not hydrogen.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein the bondattaching R^(5a) to N is in the β designation when R^(5a) is nothydrogen.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein the bondattaching R⁵ to N is in the α designation when R⁵ is not hydrogen.

According to another embodiment of the first aspect of the presentinvention are provided compounds of Formula (I) wherein the bondattaching R^(5a) to N is in the α designation when R^(5a) is nothydrogen.

Other embodiments of the first aspect of the present invention providecompounds of Formula (I) comprising two or more of the above embodimentsof the first aspect suitably combined.

Embodiments of a second aspect of the present invention provide a methodfor inhibiting tumor growth in a mammalian host which comprises theadministration to said host of a tumor-growth inhibiting amount of acompound of the present invention as defined herein.

Embodiments of a third aspect of the present invention provide a methodfor inhibiting tumor growth in a mammalian host, particularly a humanhost, comprising the administration to said host of a tumor-growthinhibiting amount of a pharmaceutical formulation of a compound of thepresent invention as defined herein.

Other embodiments and aspects of the invention will be apparentaccording to the description provided below.

DETAILED DESCRIPTION OF THE INVENTION

The description of the invention herein should be construed in congruitywith the laws and principals of chemical bonding. An embodiment oraspect which depends from another embodiment or aspect, will describeonly the variables having values and provisos that differ from theembodiment or aspect from which it depends. Thus, for example, anembodiment which reads “the compound of formula (I) according to then^(th) aspect of the invention, wherein W is C” should be read toinclude all remaining variables with values defined in the n^(th) aspectand should be read to further include all the provisos, unless otherwiseindicated, pertaining to each and every variable in the nth aspect.Where a variable is defined as having a value of zero, it is understoodthat the bond attached to said variable should be removed. For example,if n=0 and R—X—V_(n) wherein n can be 0 or 1, then it is understood thatthe structure described is R—X not R—X—.

The numbers in the subscript after the symbol “C” define the number ofcarbon atoms a particular group can contain. For example “C₁₋₇alkyl”means a straight or branched saturated carbon chain having from one toseven carbon atoms including without limitation groups such as methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl,n-pentyl, sec-pentyl, isopentyl, n-hexyl and n-heptyl. The term“halogen” includes fluoro, chloro, bromo and iodo.

It is to be understood that the present invention includes any and allpossible stereoisomers, geometric isomers, diastereoisomers,enantiomers, anomers and optical isomers, unless a particulardescription specifies otherwise.

The compounds of this invention can exist in the form ofpharmaceutically acceptable salts. Such salts include addition saltswith inorganic acids such as, for example, hydrochloric acid andsulfuric acid, and with organic acids such as, for example, acetic acid,citric acid, methanesulfonic acid, toluenesulfonic acid, tartaric acidand maleic acid. Further, in case the compounds of this inventioncontain an acidic group, the acidic group can exist in the form ofalkali metal salts such as, for example, a potassium salt and a sodiumsalt; alkaline earth metal salts such as, for example, a magnesium saltand a calcium salt; and salts with organic bases such as atriethylammonium salt and an arginine salt. The compounds of the presentinvention may be hydrated or non-hydrated.

The compounds of this invention can be administered in such oral dosageforms as tablets, capsules (each of which includes sustained release ortimed release formulations), pills, powders, granules, elixirs,tinctures, suspensions, syrups and emulsions. The compounds of thisinvention may also be administered intravenously, intraperitoneally,subcutaneously, or intramuscularly, all using dosage forms well known tothose of ordinary skill in the pharmaceutical arts. The compounds can beadministered alone, but generally will be administered with apharmaceutical carrier selected upon the basis of the chosen route ofadministration and standard pharmaceutical practice. Compounds of thisinvention can also be administered in intranasal form by topical use ofsuitable intranasal vehicles, or by transdermal routes, usingtransdermal skin patches. When compounds of this invention areadministered transdermally the dosage will be continuous throughout thedosage regimen.

One aspect of the present invention involves administration of thecompounds of the present invention, or pharmaceutically acceptable saltsor solvates thereof, to a mammal implanted with a tumor or susceptibleto cancer formation. In general the compound would be given in a doserange of from about 0.01 mg/kg to about the MTD (maximum tolerateddose). The dosage and dosage regimen and scheduling of a compounds ofthe present invention must in each case be carefully adjusted, utilizingsound professional judgment and considering the age, weight andcondition of the recipient, the route of administration and the natureand extent of the cancer disease condition. The term “systemicadministration” as used herein refers to oral sublingual, buccal,transnasal, transderrnal, rectal, intramascular, intravenous,intraventricular, intrathecal, and subcutaneous routes. In accordancewith good clinical practice, it is preferred to administer the instantcompounds at a concentration level which will produce effectivebeneficial effects without causing any harmful or untoward side effects.

Synthesis

Procedures for the preparation of compounds of the present inventioncompounds are illustrated in Schemes 1-4:

Compounds of the present invention and their methods of preparation arefurther described by the following non-limiting examples.

Synthesis of Intermediates

Several intermediate compounds as well as other conventional startingmaterials, used in the preparation of final products of compounds of thepresent invention, were generally known in the literature (WO 9807433)or were commercially available. Representative syntheses of some ofthese compounds are nevertheless provided hereinbelow.

The variables described in the above schemes have the same values asdescribed in Formula (I), except for V in the above schemes which equalsO, and W in the above schemes which equals R⁷ and R⁸ according toFormula (I). The benzyl (Bn) protecting group is illustrated as aparticular moiety to “protect” a hydroxyl functionality, but othersuitable protecting groups well known to one skilled in the art may beused in lieu of benzyl or the like. Such suitable protecting groups areadequately described in Green's Protecting Groups in Organic Synthesis(John Wiley and Sons, New York).

The starting materials in Scheme I, III and IV are glycosylatedindolopyrrolocarbazoles and their preparation is described in WO9807433.Selective derivatization at the 6′-position may be achieved directlyfrom compound of Formula 15 (R¹═H, R²═OH), wherein all sugar hydroxylgroups are unprotected. Such chemoselective activation of the6′-hydroxyl group to a good leaving group, such as mesylate or halide,is done in the presence of a base like triethylamine or pyridine usingreagents for activation of a hydroxyl group to a good leaving group,such as methanesulfonyl chloride and others typically so used by oneskillled in the art. More particular conditions are pyridine andmethanesulfonyl chloride at 0° C.

Intramolecular nucleophilic displacement of the 6′-mesylate, or othersuch leaving groups, by the 3 ′-hydroxyl moiety is catalyzed by a baselike triethylamine or Hunig's base, but more particularly fluoride ion(such as tetrabutylammonium fluoride). Typical solvents for thisreaction are, but are not limited to, DMSO, NMP, THF,N,N-dimethylimidazolidinone or DMF at temperatures from 0° C. to 150°C., but more particularly at 85° C. The product of such anintramolecular displacement of a 6′-leaving group by the nucleophilic3′-hydroxyl group is the 3′,6′-anhydrosugar derivative of theindolopyrrolocarbazole of Formula (I).

Other methods for the synthesis of 3′,6′-anhydrosugar analogs of Formula(I) from compounds of Formula 15 employ typical conditions of theMitsunobu reaction, wherein the combination of triphenylphosphine (TPP)and diisopropylazodicarboxylate (DIAD) is used to activate the6′-hydoxyl group towards intramolecular displacement by the 3′-hydroxylgroup. Typical solvents for this reaction are, but not limited to,benzene, toluene, dioxane, more particularly THF or pyridine attemperatures from −15° C. to 80° C, and more particularly roomtemperature. Other reagents and/or combinations similar to TPP and DIADmay also be employed, such as diethylazodicarboxylate (DEAD) and TPP, ortri(O)-tolylphosphine, as well as TMAD and tributylphosphine and ADDPand trimethylphosphine, as well as combinations thereof. Additives suchas 4-dimethylaminopyridine (4-DMAP) and imidazole may also be used toimprove the yield and accelerate the rate of this reaction. For example,more particular is the use of 4-dimethylaminopyridine (4-DMAP) incombination with TPP and DIAD in such solvents as THF or the like. Theuse of the additive 4-DMAP is preferred and a preferred substrate forthis modification is the glycosylated indolopyrollocarbozole of Formula20 wherin R¹═R²═H and the hydroxyl moiety at the 2′ position ispreferably protected as the benzyl ether. In such a substrate, the6′-hydroxyl moiety is activated towards intramolecular nucleophilicdisplacement by the free 3′-hydroxyl moiety, which is the only otherhydroxyl moiety present.

Another method for the synthesis of 3′,6′-anhydrosugar analogs ofFormula (I) employs as a substrate the 6′-fluoro sugar analog ofFormulas 16A or 16B. Under appropriate conditions, hydrazine or ammoniumacetate in ethanol at reflux, the 6′-fluorine can serve as a leavinggroup towards the intramolecular nucleophilic displacement by the3′-hydroxyl moiety giving products of Formula (I). These examples,wherein fluorine serves a leaving group, would not generally be expectedby one skilled in the art.

Rearrangement of the 3′,6′-anhydrosugar moiety under condiditons such asthose typically used to convert the imide nitrogen to its free NH form(from its protected precursor) or to its N-O-benzyl form (from the same)can also serve to induce a novel rearrangement to yield glycosylatedanalogs such as the bicyclo [3.3.0] analog shown in Formula 17. Ethanolcan be used as the solvent and at a reaction temperature including, butnot limited to, that achieved by reflux of the solvent.

The synthesis of 3′,4′ and 2′,3′ anhydrosugar analogs of Formulas 18-19proceed from their corresponding 3′,4′ and 2′,3′ trans-diol sugaranalogs. Methods for the synthesis of compounds of Formulas 18-19 viadehydration utilize similar conditions employed for the synthesis of the3′,6′-anhydrosugar analogs of Formula (I) (vide supra). A particularmethod for the synthesis of compounds of Formulas 18 and 19 is theMitsunobu reaction using TPP and DIAD in THF, although other reagentcombinations and solvents delineated above for the synthesis ofcompounds of Formula (I) may also be employed. A particular temperaturefor the synthesis of 3 ′,4′ and 2′,3 ′-anhydrosugar analogs of 18-19 isroom temperature to 50° C.

All anhydrous reactions were performed under an atmosphere of nitrogenor argon using either commercially available dry solvents or freshlydistilled solvents. Melting points were determined in an open capillarytube with a Thomas-Hoover melting point apparatus and are uncorrected.Column chromatography was performed using EM Science silica gel 60(230-400 mesh) with the designated solvent system as eluant. Thin-layerchromatography was done on E. Merck silica gel 60 F₂₅₄ plates (0.5 mm).HPLC purity determinations were done using either a Shimadzu LC-10ASwith a SPD-10AV UV-Vis detector and one of the folowing columns; YMCCombiscreen ODS-A (4.6×50 mm), or HP Zorbax SB-C18 (4.6×750 mm); or, anHP 1090 DR5 with a diode array detector and a Waters Nova-Pak C18 column(3.9×150 mm). Infrared spectra were recorded on a Nicolet Protégé 460FTIR as thin films or KBr pellets. ¹HNMR spectra were recorded on eithera Bruker AMX-400 or a Bruker ARX-500 NMR spectrometer and chemicalshifts are expressed in parts per million (ppm or δ) with the solvent inuse as internal standard. Coupling constants are given in hertz (Hz) andmultiplets are designated as follows; singlet (s), doublet (d), triplet(t), quartet (q), muliplet (m), and broad (br). Low resolution massspectra were determined on a Finnigan Matt TSQ-7000 triple stagequadrapole spectrometer (positive/negative ESI) operated in the negativeion mode.

Starting materials in the examples below may be synthesized by themethods disclosed in WO9807433, examples 1-106.

EXAMPLE 1

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

To a mixture of3,9-difluoro-12,13-dihydro-13-[6—O—(methylsulfonyl)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(8.0 mg, 0.013 mmol) and tetrabutylammonium fluoride trihydrate (30 mg,0.095 mmol) was added anhydrous DMF (1 mL). The resulting solution wastreated with activated powdered 4 A sieves, magnetically stirred underN₂ at room temperature for 45 min, and then heated at 85° C. for 18 h.The reaction mixture was diluted with EtOAc (300 mL), washed with water(4×75 mL) and brine (75 mL), and dried (Na₂SO₄). Evaporation in vacuo,followed by purification by flash chromatography on silica gel with 2-3%methanol in methylene chloride gave 4.8 mg (69%) of the pure titlecompound: 500 MHz COSY ¹H NMR (d₆-DMSO) δ 8.86 (d, 1H, J=9.6, 2.7 Hz),8.75 (dd, 1H, J=9.7, 2.6 Hz), 7.76 (dd, 1H, J=9.1, 4.4 Hz), 7.72 (dd,1H, J=8.9, 4.5 Hz), 7.48 (ddd, 1H, J=9.1, 9.0, 2.7 Hz), 7.44 (ddd, 1H,J=9.0, 8.9, 2.8 Hz), 7.00 (d, 1H, J=7.7 Hz, 1′H), 4.58-4.55 (m, 2H,3′,6′H), 4.45 (d, 1H, J=7.7 Hz, 2′H), 4.16 (d, 1H, J=5.2 Hz, 6″ H), 4.10(d, 1H, J=9.9 Hz, 5′H), 4.01-3.99 (d, 1H, 4′H); FAB mass spectrum, m/e506 (M⁺).

EXAMPLE 2

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-α-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

To a stirred solution of3,9-Difluoro-12,13-dihydro-13-[(6-fluoro)-α-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-[4-(t-butyl)benzyl]-)-dione in absolute ethanol (150mL) was added 4.45 M KOH (55 mL). The resulting blood red solution washeated to reflux until all the ethanol was boiled off and a red solidgummed out. The reaction was cooled to room temperature, treated with 1N HCl (25 mL), and solid ammonium acetate (100 g) was added. Theresulting suspension was heated to reflux for 18 h and the volume wasconcentrated by about two thirds. The resulting suspension was partionedwith ethyl acetate and concentrated HCl. The orgainc layer was washedwith water, sodium bicarbonate, and brine, and dried (Na₂SO₄).Evaporation in vacuo followed by flash column chromatography on silicagel using an acetone/methylene chloride gradient gave the titlecompound: 500 MHz COSY ¹H NMR (d₆-DMSO) δ 8.86 (d, 1H, J=9.6, 2.7 Hz),8.75 (dd, 1H, J=9.7, 2.6 Hz), 7.76 (dd, 1H, J=9.1, 4.4 Hz), 7.72 (dd,1H, J=8.9, 4.5 Hz), 7.48 (ddd, 1H, J=9.1, 9.0, 2.7 Hz), 7.44 (ddd, 1H,J=9.0, 8.9, 2.8 Hz), 7.00 (d, 1H, J=7.7 Hz, 1′H), 4.58-4.55 (m, 2H,3′,6′H), 4.45 (d, 1H, J=7.7 Hz, 2′H), 4.16 (d, 1H, J=5.2 Hz, 6″H), 4.10(d, 1H, J=9.9 Hz, 5′H), 4.01-3.99 (d,1H, 4′H); ESI (NEG) mass spectrum,m/e 504 (M−H)⁻.

EXAMPLE 3

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-hydroxyl)-dione:

To a solution of3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-O-benzyl)-dione:(10 mg) in methanol was added platinum (IV) oxide. The resultingsuspension was treated with hydrogen at 70 PSI on a Parr shaker for 18h. The catalyst was filtered thru a small pad of celite and the solventremoved in vacuo. Purification on Sephadex LH-20 with methanol elutiongave 7.1 mg of the title compound: 300 MHz ¹H NMR (d₆-Acetone) δ8.90-8.70 (m, 2H), 7.95-7.20 (m, 4H), 6.62 (d, 1H, J=7.5 Hz), 5.20-4.05(m, 6H); ESI (NEG) mass spectrum, m/e 520 (M−H)^(−.)

EXAMPLE 4

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-β-D-glucopyranosyl]-5H-indolo[2,3-4]pyrrolo[3,4-c]arbazole-5,7(6-N-amino)-dione:

To a stirred solution of3,9-Difluoro-12,13-dihydro-13-[(6-fluoro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-[4-(t-butyl)benzyl]-)-dione (150 mg) inabsolute ethanol (250 mL) was added 4.45 M KOH (50 mL). The resultingblood red solution was heated to reflux until all of the ethanol boiledoff and a red solid gummed out. The reaction was cooled to roomtemperature and treated with conc. HCl (51 mL). Hydrazine (75 g) andadditionl ethanol (250 mL) were added and the reaction was allowed toreflux for 5 days. The reaction volume was concentrated by approximately⅔ volume, cooled to room temperature, and partitioned with ethyl acetateand water. The orgainc layer was washed with water, sodium bicarbonate,and brine, and dried (Na₂SO₄). Rotary evaporation followed bypurification on Sephadex LH-20 chromatography in methanol gave the titlecompound: 500 MHz COSY ¹H NMR (d₆-DMSO) δ 8.90 (dd, 1H), 8.79 (dd, 1H),8.05 (dd, 1H), 7.81 (dd, 1H), 7.53-7.43 (m, 2H), 6.69 (d, 1H, J=7.9 Hz,1′H), 5.01 (s, OH), 5.01-4.93 (dd, 1H, 4′H), 4.60 (brs, 1H, 5′H), 4.50(brs, 1H, 3′H), 4.17 (brs, 1H, 2′H), 3.99-3.95 (m, 2H, 6′H, 6″H); FABmass spectrum, m/e 520 (M⁺).

EXAMPLE 5

3,9-Difluoro-12,13-dihydro-13-[(2,6-dihydroxy)-(4,8-dioxa)-bicyclo[3.3.0]oct-3-yl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-O-benzyl)-dione:

To a stirred solution of3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-[4-(t-butyl)benzyl]-)-dione:(22.5 mg) in absolute ethanol (28 mL) was added 4.45 M KOH (7 mL). Theresulting blood red solution was heated to reflux until all of theethanol boiled off and a red solid gummed out. The reaction was cooledto room temperature and treated with conc. HCl (3 mL).O-benzylhydroxylamine hydrochloride (10 g) and additionl ethanol (20 mL)were added and the reaction was allowed to reflux overnight. Thereaction volume was concentrated by approximately ½ volume, cooled toroom temperature, and partitioned with ethyl acetate and water. Theorganic layer was washed with water, and brine, and dried (Na₂SO₄).Rotary evaporation followed by purification by flash chromatography onsilica gel using a methylene chloride/ ethyl acetate gradient gave 8.7mg of the title compound as an orange solid: 500 MHz COSY ¹H NMR(d₆-DMSO) δ 8.81 (dd, 1H, J=2.6, 9.5 Hz, H-8), 8.72 (dd, 1H, J=2.6, 9.7Hz, H-4), 8.06 (dd, 1H, J=4.3, 9.3 Hz, H-11), 7.82 (dd, 1H, J=4.6, 9.0Hz, H-1), 7.64-7.35 (m, 7H), 6.72 (d, 1H, J=4.0 Hz, 5′OH), 6.65 (d, 1H,J=8.0 Hz, 1′H), 5.67 (d, 1H, J=5.6 Hz, 2′OH), 5.32 (s, 2H, O—CH₂-Ph),4.98 (t, 1H, J_(3′,4′)=6.2 Hz, J_(4′, 5′)=6.2 Hz, 4′H), 4.62 (ddd 1H,J₄′,₅=6.2 Hz, J₅′,₆=4.6 Hz, J₅′,OH =4.0 Hz, 5′H), 4.50 (dd, 1H,J_(2′,3′)=3.8 Hz, J_(3′,4′)=6.2 Hz, 3′H), 4.13 (ddd, 1H, J_(1′,2′)=8.0Hz, J_(2′,3′)=3.8 Hz, J_(2′,OH)=5.6 Hz, 2′H), 3.98 (d, 2H, J_(5′,6′)=4.6Hz, J_(5′,6″)=0 Hz, 6′H, 6″H); FAB mass spectrum, m/e 611 (M⁺); ESI(NEG) mass spectrum, m/e 610 (M−H)⁻.

EXAMPLE 6

3,9-Difluoro-12,13-dihydro-13-[(2,6-dihydroxy)-(4,8-dioxa)-bicyclo[3.3.0]oct-3-yl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-hydroxyl)-dione:

To a solution of the product from Example 5 (6.4 mg) in 1:1methanol:ethyl acetate (3.5 mL) was added 20% palladium hydroxide/C and10% palladium/C. The resulting suspension was treated with hydrogen at75 PSI on a Parr shaker for 18 h. The catalyst was filtered thru a smallpad of celite and the solvent removed in vacuo. Purification on SephadexLH-20 with methanol elution gave 3.6 mg of the title compound: 500 MHzCOSY ¹H NMR (d₆-DMSO) δ 8.77 (dd, 1H), 8.59 (dd, 1H), 7.99 (dd, 1H),7.72 (dd, 1H), 7.45-7.25 (m, 2H), 6.87 (brs, 1H), 4.93-4.84 (m, 1H,),4.58-4.43 (m, 2H), 4.34 (brs, 1H), 4.08-3.88 (m, 2H); FAB mass spectrum,m/e 521 (M⁺).

EXAMPLE 7

3,9-Difluoro-12,13-dihydro-13-[(2,3-anhydro)-(4,6-difluoro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-]carbazole-5,7(6H)-dione:

To a stirred solution of3,9-Difluoro-12,13-dihydro-13-[(4,6-difluoro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(23.7 mg, 0.045 mmol) and triphenylphosphine (36.6 mg, 0.140 mmol) inanhydrous THF (1.0 mL) was added diisoproply azodicarboxylate (27 μL,0.137 mmol). The reaxtion was allowed to stir at room temperature for 1h, then warmed to 50° C. for 3 h. Additional triphenylphosphine (39 mg,0.149 mmol) and diisoproply azodicarboxylate (29 μL, 0.148 mmol) wereadded and the resulting red solution was stirred at 50° C. overnight.The reaction was quenched with water (1 drop) and the solvent removed invacuo. Purification on Sephadex LH-20 gave 2.0 mg of the title compound:500 MHz COSY ¹H NMR (d₆-DMSO) δ 8.88 (dd, 4H), 8.77 (dd,1H), 8.16-8.05(m, 1H), 7.81-7.69 (m, 1H), 7.43 (brs, 1H, 1′H), 5.11 (dd, 2H, 4′H,4″H), 4.76 (s, 1H, 6″H), 4.67 (s, 1H, 6′H), 4.52-4.36 (m, 1H, 5′H),4.06-3.91 (m, 2H, 3′H, 2′H); ESI (NEG) mass spectrum, m/e 508 (M−H)⁻.

EXAMPLE 8

3,9-Difluoro-12,13-dihydro-13-[(3,4-anhydro)-β-D-glucopyranosyl]-5H-indolo [2,3 a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

To a stirred solution of3,9-Difluoro-12,13-dihydro-13-[β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(240 mg, 0.495 mmol) and triphenylphosphine (263 mg, 1.00 mmol) inanhydrous THF (10 mL) was added duisoproply azodicarboxylate (200 μL,1.01 mmol). The resulting red solution was allowed to stir at roomtemperature for 2 h, after which is was quenched with water (5 drops)and evaporated to dryness in vacuo. Flash column chromatography onsilica gel using an acetone:hexane gradient followed by Sephadex LH-20purification in methanol gave 43.4 mg (23%) of the title compound: 500MHz COSY ¹H NMR (d₆-DMSO) δ 11.22 (brs, 1H), 8.85 (dd, 1H), 8.76 (dd,1H), 7.97 (brs, 1H), 7.65 (brs, 1H), 7.58-7.39 (m, 2H), 6.21 (d, 1H,1′H), 5.88 (d, 1H, 2′OH), 5.40 (brs, 1H, 6′OH), 4.71 (brs, 1H, 5′H),4.12 (brs, 1H, 2′H), 4.01-3.82 (m, 2H, 6′H, 6″H), 3.74 (brs, 1H, 4′H),3.44 (d, 1H, 3′H); ESI (NEG) mass spectrum, m/e 504 (M−H)⁻.

EXAMPLE 9

3,9-Difluoro-12,13-dihydro-13-[(2—O-benzyl)-(4-dexoy)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

A solution of zinc chloride (6.5 g, 47.7 mmol) in 2:1 aceticanhydride/acetic acid (48 mL) was added to a suspension of3,9-Difluoro-12,13-dihydro-13-[2,3,6-(O-benzyl)-(4-deoxy)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-[4-(t-butyl)benzyl]-)-dione (5.0 g, 5.41 mmol)in 2:1 acetic anhydride/acetic acid (120 mL). The reaction mixture washeated to 65° C. for 22 h, cooled to room temperature, and diluted withethyl acetate. The organic layer was washed with water (3×200 mL),sodium bicarbonate (3×200 mL), water (2×200 mL), and brine (2×200 mL),and dried (Na₂SO₄). Evaporation in vacuo gave a crude product which wasdeblocked according to the procedure described in WO9807433 to give 1.38g (43%) of the title compound: 300 MHz ¹H NMR (d₆-DMSO) δ 11.75 (brs,1H), 11.25 (brs, 1H), 8.85 (dd, 1H, J=9.6, 3.1 Hz), 8.77 (dd, 1H, J=9.8,2.8 Hz), 8.00 (dd, 1H, J=9.6, 4.4 Hz), 7.70 (dd, 1H, J=8.8, 4.6 Hz),7.53-7.43 (m, 2H), 6.90 (t, 1H, J=7.7 Hz), 6.77 (dd, 2H, 7.7, 7.1 Hz),6.37 (d, 1H, 9.1 Hz), 6.14 (t, 1H), 6.08 (d, 2H, J=7.1 Hz), 5.28 (d, 1H,5.8 Hz), 4.18-3.64 (m, 5H), 3.21-3.17 (m, 2H), 2.42-2.28 (m, 1H),2.07-1.98 (m, 1H); ESI (NEG) mass spectrum, m/e 596 (M−H)⁻.

EXAMPLE 10

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-(2—O-benzyl)-(4-deoxy)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

To a 5-10° C. solution of the product from Example 9 (1.03 g, 1.72mmol), triphenyl phosphine (1.82 g, 6.93 mmol) and4-dimethylaminopyridine (539 mg, 4.41 mmol) in anhydrous THF (95 mL) wasadded diisopropyl azodicarboxylate (1.37 mL, 6.96 mmol). The resultingred solution was stirred at room temperature for 90 minutes, cooled to0° C., quenched with water (3.5 mL) and the solvent removed in vacuo.The resulting residue was evaporated from absolute ethanol (100 mL),redissolved in a mixture of THF and methylene chloride and applied to aflash column packed in methylene chloride. The column was eluted with agradient from methylene chloride to 5% ethyl acetate in methylenechloride to give 693 mg (69%) of the title compound: 500 MHz COSY ¹H NMR(d₆-DMSO) δ 12.0 (brs, 1H), 11.27 (s, 1H), 8.92-8.84 (m, 2H), 7.84-7.72(m, 2H), 7.55-7.50 (ddd, 1H), 7.45-7.37 (ddd, 1H), 6.86 (t, 1H), 6.62(t, 2H), 6.44 (d, 1H, J=7.35 Hz, 1′H), 6.34 (d, 2H), 5.00 (brs, 1H,5′H), 4.65 (d, 1H, J=5.7 Hz, 3′H), 4.33 (d, 1H, J=12.4 Hz, 2′-CH₂Ph),4.21 (d, 1H, J=9.2 Hz, 6′H), 4.10-4.00 (m, 2H, 2′H and 2′-CH₂Ph), 3.81(d, 1H, J=9.2 Hz, 6″H), 3.35-3.25 (m, 1H, 4′H), 2.08-1.99 (m, 1H, 4″H);ESI (NEG) mass spectrum, m/e 578 (M−H)⁻.

EXAMPLE 11

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro-4-deoxy)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

To a solution of the product from Example 10, (733 mg, 1.26 mmol) in 3:1methanol/THF (400 mL) was added 30% palladium on carbon (510 mg) and 20%palladium hydroxide on carbon (1.25 g). The resulting suspension wasflushed with nitrogen, then with hydrogen and allowed to stir overnightunder a hydrogen atmosphere. The reaction was filtered thru a small padof Celite, the catalyst washed with THF and methanol, and the filtrateevaporated in vacuo. The resulting residue was evaporated onto silicagel, applied to a flash column packed in 10% acetone/methylene chloride,and the column was eluted with a gradient from 10% acetone/methylenechloride to 80% acetone/methylene chloride. Further purification onSephadex LH-20 in acetone gave 299 mg (49%) of the title compound: 500MHz COSY ¹H NMR (d₆-DMSO) δ 11.99 (brs, 1H), 11.26 (s, 1H), 9.00-8.96(dd, 1H), 8.90-8.85 (dd, 1H), 7.95-7.82 (m, 2H), 7.62-7.47 (m, 2H), 6.33(d, 1H, J=7.20 Hz, 1′H), 5.76 (d, 1H, J=5.10 Hz, 2′OH), 4.99 (brs, 1H,5′H), 4.40 (d, 1H, J=5.80, 3′H), 4.36-4.32 (m, 1H, 2′H), 4.22 (d, 1H,J=8.9 Hz, 6′H), 3.83 (d, 1H, J=8.9 Hz, 6″H), 3.27 (d, 1H, J=9.65 Hz,4′H), 2.06-1.99 (m, 1H, 4″H); ESI (NEG) mass spectrum, m/e 488 (M−H)⁻.

EXAMPLE 12

2,10-Difluoro-12,13-dihydro-12-[(3,4-anhydro)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

Diisopropyl azodicarboxylate (64 μL, 0.32 mmol) was added dropwise to acold (0° C.) solution of2,10-difluoro-12,13-dihydro-12-[β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(0.15 g, 0.29 mmol) and triphenylphosphine (83 mg, 0.32 mmol) undernitrogen. The mixture was allowed to gradually warm up to roomtemperature, stirred for 16 h, diluted with ethyl acetate and washedwith saturated sodium bicarbonate solution and brine. Following dryingand solvent concentration, the residue was purified by flashchromatography on silica gel (elution with 10% methanol in chloroform)to furnish2,10-difluoro-12,13-dihydro-12,13-[1,6-anhydro-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(8.7 mg, 12%) as a yellow solid and the title compound (75.7 mg, 52%)also as a yellow solid, m.p. >300° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 11.28(s, 1H), 11.20 (s, 1H), 9.12 (dd, J=8.5, 6.0 Hz, 1H), 9.04 (dd, J=7.2,5.8 Hz, 1H), 7.83 (d, J=9.2 Hz, 1H), 7.37 (d, J=8.9 Hz, 1H), 7.29-7.21(m, 2H), 6.18 (d, J=8.2 Hz, 1H), 5.90 (d, J=6.4 Hz, 1H), 5.48 (br s,1H), 4.71-4.69 (m, 1H), 4.08 (m, 1H), 3.96 (m, 1H), 3.89-3.88 (m, 1H),3.73 (s, 1H), 3.42 (d, J=3.8 Hz, 1H); IR (KBr, cm⁻¹) 3458, 3368, 2929,1747, 1698, 1624, 1578, 1452,1406, 1385, 1330, 1232, 1171, 1115,1061,919,836,762; MS (-ESI, M−H⁻) m/z 504.

EXAMPLE 13

3,9-Difluoro-13-(3,6-anhydro-α-D-glucopyranosyl)-5H,13H-benzo[β]thienyl[2,3-a]pyrrolo[3,4-c] carbazole-5,7(6H-dione:

Diisopropyl azodicarboxyl ate (2.2 mL, 11.21 mmol) was added dropwise toa cold (0° C.) suspension of 3,9-difluoro-6-methyl-5H, 13H-benzo[β]thienyl[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (2.0 g, 5.10 mmol),triphenylphosphine (2.94 g, 11.21 mmol)and6-fluoro-2,3,4-tribenzyl-D-glucopyranose (3.46 g, 7.64 mmol) undernitrogen. The mixture was allowed to gradually warm up to roomtemperature where it stirred for 2 h before it was cooled to 0° C. andtreated with additional 6-fluoro-2,3,4-tribenzyl-D-glucopyranose (1.73g, 3.82 mmol), triphenylphosphine (1.47 g, 5.61 mmol) and DIAD (1.1 mL,5.61 mmol). After stirring for an additional 1 h at room temperature,the mixture was diluted with ethyl acetate and washed with water andbrine. Following drying and solvent concentration, the residue waspurified by flash chromatography on silica gel (gradient elution with10% ethyl acetate in hexane followed by 15% and finally with ethylacetate/tetrahydrofuran/hexane (15/5/80) to furnish the coupled productas a yellow foam which was carried on directly. The substrate was takenup in 95% ethanol (50 mL) and subjected to transfer hydrogenation with20% palladium hydroxide on carbon (1.5 g) and cyclohexene (40 mL). Themixture was refluxed for 7 h before additional catalyst (1.5 g),cyclohexene (40 mL) and ethanol (50 mL) were added. After an additional16 h at reflux, the mixture was filtered hot through Celite was washedwith THF and methanol. The filtrate was concentrated down in vacuo.Purification of the residue by flash chromatography on silica gel(elution with 10% methanol in chloroform) yielded the debenzylatedproduct as a yellow solid which was taken on further. Potassiumhydroxide (5M, 10 mL) was added to a stirred suspension of thedebenzylated product in absolute ethanol (2 mL) at room temperature. Themixture was stirred at room temperature for 2 d before it was heated to50° C. and sparged with air in order to remove most of the ethanol.After cooling for 15 min at 0° C., concentrated hydrochloric acid wasadded in portions until a precipitate formed and remained (pH=1). Thissuspension was stirred at room temperature for 24 h before it wasdiluted with ethyl acetate and tetrahydrofuran and washed with 1N HCl,brine, dried and concentrated. Solid ammonium acetate (10 g, xs) wasadded to the residue and the mixture was fused at 120° C. was 2 h beforeit was cooled to room temperature, diluted with ethyl acetate and THFand washed with saturated sodium bicarbonate solution until a pH=9 wasachieved. Solid sodium carbonate was initially used in order to quenchmost of the acetic acid. The organic layer was then separated, washedwith brine, dried and concentrated. Purification of the residue by flashchromatography on silica gel (gradient elution with 10% methanol inchloroform followed by 15% and finally 20% methanol in chloroform)afforded a three component inseparable mixture which was furtherpurified by LH-20 chromatography (methanol, 0.3 mL/min, 36 h) to furnish3,9-difluoro-13-(6-fluoro-6-deoxy-β-D-glucopyranosyl)-5H,13H-benzo[β]thienyl[2,3-a]pyrrolo[3,4-c] carbazole-5,7(6H)-dione (226.1mg, 8.2%, 4 steps),3,9-difluoro-6-methyl-13-(6-fluoro-6-deoxy-β-D-glucopyranosyl)-5H,13H-benzo[p]thienyl[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (20.1 mg, 0.7%, 4steps), and the title compound (10.1 mg, 0.4%, 4 steps) as a yellowsolid, m.p. >305° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 11.50 (v br s, 0.7H),9.54 (d, J=11.4 Hz, 1H), 8.75 (dd, J=9.7, 2.5 Hz, 1H), 8.46 (dd, J=9.2,4.8 Hz, 1H), 8.11 (dd, J=8.7, 5.3 Hz, 1H), 7.40-7.32 (m, 2H), 7.00(d,J=3.1, 1H), 4.63 (s, 1H), 4.42-4.39 (m, 2H), 4.31-4.29 (m, 1H),4.31-4.29 (m, 1H), 4.20 (d, J=9.0 Hz, 1H), 4.11 (s, 1H), -0.08 (br s,2H); IR (KBr, cm⁻¹) 3341, 3184, 2961, 1756, 1699, 1620, 1604,1573, 1476,1457, 1424, 1328, 1259, 1200, 1165, 1115, 1101,1081,1062,1019,920,878,810,762;MS(-ESI,M−H⁻)m/z 521, (+ESI, M+H⁺) m/z 523.

EXAMPLE 14

12-(3,6-anhydro-β-D-glucopyranosyl)-2,10-difluoro-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

2,10-Difluoro-12,13-dihydro-12-[β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(1.0 g, 1.91 mmol) was dissolved in dry pyridine under nitrogen at roomtemperature and treated with flame-dried, powdered 4A molecular sieves(0.60 g). The mixture. was cooled to −20° C. for 15 min beforemethanesulfonyl chloride (0.26 mL, 3.34 mmol, 1.75 eq.) was added neat.The flask was sealed and stored at 0° C. for 6 h prior to concentrationin vacuo. Purification of the residue by flash chromatography on silicagel (elution with tetrahydrofuran/dichloromethane/methanol, 68:30:2)gave an enriched fraction (280 mg, 24%) containing the title compound aswell as a small amount of other closely-spaced by-products which wascarried on directly. Potassium phthalimide (0.45 g, 2.43 mmol) was addedin one portion to a stirred solution of2,10-difluoro-12,13-dihydro-12-[6—O-(methylsulfonyl)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(200 mg, 0.33 mmol) in anhydrous dimethylformamide (10 mL) before themixture was heated to 130° C. for 3 h, cooled to ambient temperature,and concentrated down in vacuo overnight. The residue was then taken upin ethyl acetate (some tetrahydrofuran was added) and washed with 0.1Nhydrochloric acid and brine. Following drying and solvent concentration,the residue was purified by flash chromatography on silica gel (elutionwith 7% methanol in chloroform) to yield2,10-difluoro-12,13-dihydro-12-[6-deoxy-6-(phthalimido)-β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione(46 mg, 21%) as a yellow solid as well as the title compound (22 mg,13%, 2 steps) also as a yellow solid.

For title compound: M.p. >300° C.: ¹H NMR (500 MHz, DMSO-d₆) δ 11.21 (s,1H), 11.10 (br s, 1H), 9.15 (dd, J=8.7, 5.9 Hz, 1H), 9.05 (dd, J=9.0,5.8 Hz, 1H), 7.47 (d, J=9.4 Hz, 1H), 7.42 (d, J=10.2 Hz, 1H), 7.31 (dt,J=9.0, 2.2 Hz, 1H), 7.24 (dt, J=9.2, 2.2 Hz, 1H), 6.97 (d, J=7.7 Hz,1H), 6.39 (br s, 1H), 5.87 (br s, 1H), 4.60 (s, 2H), 4.44 (d, J=7.2 Hz,1H), 4.17 (m, 1H), 4.07-4.02 (m, 2H); IR (KBr, cm⁻¹) 3414, 1747, 1700,1623, 1580, 1452, 1386, 1327, 1230, 1167, 1114, 1055, 1021,760; HRMS(neg ESI, M−H⁻) calc'd for C₂₆H₁₆F₂N₃O₆ 505.1086, obsd 504.1029.

EXAMPLE 15

2,3,9,10-Tetrafluoro-12-(2—O-benzyl-3,6-anhydro-4-deoxy-β-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione:

To a solution of2,3,9,10-tetrafluoro-12-(2—O-benzyl-4-deoxy-β-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione(0.018 g, 0.028 mmol) in 0.5 mL of dry pyridine was added a solution oftriphenylphosphine (0.022 mg, 0.084 mmol) in 0.1 mL of dry pyridine andthen diisopropyl azodicarboxylate (DIAD) (0.017 mL, 0.084 mmol) wasadded dropwise at room temperature under Ar. The resulting blood-redmixture was stirred at room temperature under Ar for 16 h and then itwas quenched by adding water (0.1 mL), followed by methanol (0.1 mL).This mixture was evaporated in vacuo and the residue was purified usingpreparative tlc (4×20×20 cm plates, 0.5 mm SiO₂/ CH₂Cl₂-MeCN, 9:1) togive the title compound (0.007 g, 41%) as a bright yellow solid: ¹H NMR(THF-d₈, 400 MHz) δ.10.83 (br s, 1H), 10.08 (br s, 1H), 9.12 (dd,J=11.0, 8.6 Hz, 1H), 9.02 (dd, J=11.0, 8.4 Hz, 1H), 7.72 (m, 1H), 7.36(m, 1H), 6.83 (t, J=7.4 Hz, 1H), 6.70 (t, J=7.6 Hz, 2H), 6.59 (d, J=7.6Hz, 2H), 6.23 (d, J=7.4 Hz, 1H), 4.93(s, 1H), 4.76 (d, J=6.0 Hz, 1H),4.41 (d, J=11.7 Hz, 1H), 4.31 (d, J=8.9 Hz, 1H), 4.29 (d, J=10.2 Hz,1H), 4.13 (d, J=11.7 Hz, 1H), 3.87 (d, J=9.2 Hz, 1H), 3.10 (d, J=13.4Hz, 1H), 2.10 (dd, J=13.4, 6.0 Hz); MS (ESI⁻) m/e 614 (M−H)⁻.

EXAMPLE 16

2,3,9,10-Tetrafluoro-12-(3,6-anhydro-4-deoxy-β-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione:

A mixture of 2,3,9,10-tetrafluoro-12-(2—O-benzyl-3,6-anhydro-4-deoxy-β-D-glucopyranosyl)-6, 7, 12,13-tetrahydro(5H)indolo[2, 3-a]pyrrolo[3, 4-c]carbazole-5, 7-dione(0.034 g, 0.055 mmol) and 20% Pd(OH)₂/C (0.048 g) in 5 mL of freshlydistilled THF was hydrogenated (balloon pressure) at room temperaturefor 5 days. The resulting mixture was filtered through a bed of Celiteand the cake was washed with THF and then with methanol. The filtratewas evaporated and the residue was chromatographed (Sephadex LH-20/methanol) to give the title compound (0.017 g, 59%) as a yellow solid:¹H NMR (THF-d₈, 400 MHz) δ 11.15 (br s, 1H), 10.17 (br s, 1H), 9.19 (dd,J=11.1, 8.5 Hz, 1H), 9.09 (dd, J=11.2, 8.3 Hz, 1H), 7.81 (dd, J=11.6,6.7 Hz, 1H), 7.51 (dd, 10.5, 6.7 Hz, 1H), 6.21 (d, J=7.3 Hz, 1H), 5.37(br s, 1H), 4.89 (s, 1H), 4.57 (m, 2H), 4.27 (d, J=9.7 Hz, 1H), 3.85 (d,J=9.1 Hz, 1H), 3.09 (d, J=13.4 Hz, 1H), 2.09 (dd, J=13.1, 6.0 Hz, 1H);MS (ESI⁻) m/e 524 (M−H)⁻; HPLC: 99.0% (270 nm).

EXAMPLE 17

2,3,9,10-Tetrafluoro-12-(3, 6-anhydro-4-deoxy-4,4-difluoro-β-D-glucopyranosyl)-6,7,12,13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5, 7-dione:

To a solution of 2,3,9,10-tetrafluoro-12-(4-deoxy-4,4-difluoro-β-D-glucopyranosyl)-6, 7, 12, 13-tetrahydro(5H)indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5, 7-dione (0.035 g, 0.060 mmol) in 4 mL ofdry THF was added triphenylphosphine (0.048 g, 0.18 mmol) and DIAD(0.035 mL, 0.18 mmol), at room temperature under Ar. The resultingreddish-orange solution was stirred at room temperature for 18 h andthen it was partitioned with ethyl acetate-water. The organic phase wasseparated, washed (brine), dried (Na₂SO₄) and evaporated to give ayellow gum. This residue was purified by prep. tlc (20×20 cm×0.5 mmSiO₂/THF-hexane, 1:1) and the major fraction was repurified by prep.hplc to give the title compound (0.009 g, 29%) as a yellow solid: ¹H NMR(acetone-d₆, 400 MHz) δ 10.67 (s, 1H), 9.83 (s, 1H), 8.88 (m, 1H), 8.73(m, 1H), 7.56 (m, 2H), 6.44 (m, 1H), 4.91 (m, 2H), 4.47 (d, J=10.0 Hz,1H), 4.41(d, J=11.0 Hz, 1H), 4.27 (m, 1H); MS (ESI⁻) m/e 560 (M−H)-;HPLC: 91.1% (320 nm).

EXAMPLE 18

3,9-Difluoro-12,13-dihydro-13-[(3,6-anhydro)-β-D-galactopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione:

(Diethylamino)sulfur trifluoride (1.02 ml, 7.72 mmol) was added to astirred suspension of3,9-difluoro-12,13-dihydro-13-[β-D-glucopyranosyl]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6-N-(4-t-butyl)benzyl)-dione(1.01 g, 1.51 mmol) in 50 ml anhydrous CH₂Cl₂ at −50° C. and thereaction mixture stirred for 3.5 hours during while temperature rose to+15° C. The mixture was cooled to −78° C., quenched by addition of 5 mlCH₃OH and poured on 1N HCl. The crude product was extracted with ethylacetate, the organic layers were washed with saturated aqueousNaHCO₃-solution and brine and dried over Na₂SO₄ and concentrated invacuo.

The resulting crude product was dissolved in 100 ml Ethanol and 10 ml3.83 M aqueous KOH was added. The mixture was stirred at roomtemperature for 17 hours under argon after which 10 ml conc. aqueous HClwas added and stirring continued for an additional 30 minutes. To thiswas added 150 g NH₄OAc and an additional 200 ml ethanol and the mixtureheated to reflux for 3 days. The reaction was poured on saturatedaqueous NaHCO₃-solution and extracted with ethyl acetate. The organiclayers were washed with water and brine and dried over Na₂SO₄ andconcentrated in vacuo.

Flash column chromatography on silica gel using a gradient from 100%chloroform to 4% methanol in chloroform followed by Sephadex LH-20purification in methanol gave 22.0 mg of the title compound: 500 MHz¹H-NMR (d₆-DMSO) δ 12.76 (bs, 1H), 11.15 (bs, 1H), 8.90 (dd, J=2.6, 9.6Hz, 1H), 8.75 (dd, J=2.6, 9.8 Hz, 1H), 7.95 (m, 1H), 7.67 (m, 1H), 7.44(m, 2H), 6.32 (d, J=8.3 Hz, 1H), 5.35 (d, J=4.6 Hz, 1H), 4.68 (s, 1H),4.60 (m, 1H), 4.58 (m, 2H), 4.41 (d, J=8 Hz, 1H), 4.28 (d, J=5.6 Hz,1H), 4.16 (d, J=9.3 Hz, 1H); IR (KBr, cm⁻¹) 3616, 3444, 3246, 2995,1747, 1698, 1619, 1587, 1481, 1395, 1328, 1290, 1246, 1189; ESI (NEG)mass spectrum, m/e 504 (M−H⁻); HRMS (FAB, M+H⁺) m/Z_((obs.))=506.11676m/z_((calc.))=506.116748.

The title compound represents one of the major relevant productsisolated from this reaction sequence.

Biological Activity

The compounds of the present invention are useful pharmacologic agentswith anti-tumor properties. With topoisomerase I active properties, thecompounds can be useful as anti-tumor agents. In recent years, numerousreports have appeared in the literature suggesting that the role oftopoisomerase I targeting drugs is to stabilize a covalentDNA-topoisomerase I complex to yield enzyme-linked DNA single-strandbreaks. From a pharmacologic standpoint, there are advantages to targetTopoisomerase I. First, its occurrence at relatively high levels in bothproliferating and quiescent cells suggests that its function may beindependent of cellular growth rate. Second, topoisomerase I activeagents may be effective in slow-growing as well as rapidly proliferatingtumors. Cells from colon tumors have been shown to contain higherintracellular levels of topoisomerase I than normal mucosal cells,suggesting the possibility for a selective cytotoxic advantage. Thus,inhibition of proliferation of tumor cells by compounds of the presentinvention compounds was initially demonstrated by effective inhibitionof human topoisomerase I. Selected compounds of the present invention,usually having EC₅₀ values less than 10 μM in the topoisomerase I assay,were also tested in an inhibition of human/mouse tumor cellproliferation assay.

Topoisomerase I Activity (In Vitro)

Topoisomerase I activity was measured as described below: The procedurefor assaying compound-induced, topoisomerase I-mediated single strandbreaks in DNA was essentially that described by Hsiang, et al., (J.Biol. Chem. 1985, 260,14873-14878). Samples dissolved in 100% DMSO aseither 10 μM or 10 mg/ml solutions, unless otherwise stated, werediluted in Tris-EDTA buffer. Marine bacteriophage PM2 DNA (BoehringerMannheim) was also diluted in Tris-EDTA buffer to a concentration of0.02 μg/μl. Different dilutions of compound being evaluated were mixedwith diluted DNA and this mixture was added to 1000 unit (one unit ofenzyme activity is defined as the amount capable of relaxing 100 ng ofsupercoiled DNA in approximately 30 minutes at 37° C.) aliquots ofpurified human topoisomerase I (Topogen) in 2× reaction buffer to startthe reaction. The compound—DNA—enzyme mixture was incubated for 30minutes at 37° C. before stopping the reaction with warm stop buffercontaining sodium dodecyl sulfate and proteinase K (Sigma). Thesemixtures were allowed to incubate at 37° C. for another 10 minutes, atwhich time the mixtures were removed from the waterbath and extractedwith a 24:1 mixture of chloroform/isoamyl alcohol. Followingcentrifugation, aliquots of the aqueous phases were placed in wells of a0.9% agarose (SeaKem) gel in Tris-borate buffer containing 0.5 μg/ml ofethidium bromide and subjected to electrophoresis for 15 hours toseparate the different topological isomers and nicked and broken DNAs.After destaining the gel in water, the ethidium bromide stained DNAreaction products were visualized by exposing the gel to UV irradiation.Negatives of photographs of the irradiated gels were scanned with adensitometer and areas under the peaks were calculated in order toobtain percent single strand DNA break formation for each sample. Amedian effective concentration(EC₅₀) was obtained for each compound byinterpolation between points of the resulting dose-effect curve whichdefines the potency of the compound for its effect in inducingtopoisomerase I-mediated single strand breaks in DNA.

The topoisomerase I activity for selected compounds of the presentinvention is shown below in Table I.

TABLE I In Vitro Cell-Based Cytotoxicity Activity Example TOPO I ExampleTOPO I No. EC₅₀ (μM) No. EC₅₀ (μM) 2 0.36 13 0.11 1 0.085 14 5.50 180.18 7 0.58 4 0.01 8 0.018 3 0.014 12 0.58 11 0.13 6 0.01 17 15.0

The proliferation inhibition activity against murine P388 cell line wasmeasured as follows. Cytotoxicity was assessed in HCT116 human coloncarcinoma cells by XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide assay as described in the literature by Scudiero, D A,Shoemaker, R H, Paull, K D, Monks, A, Tierney, S, Nofziger, T H,Currens, M J, Seniff, D, and Boyd, M R. Evaluation of a solubletetrazolium/formazan assay for cell growth and drug sensitivity inculture using human and other tumor cell lines, was done according tothe procedure described in Cancer Res. 48: 4827-4833, 1988. Cells wereplated at 4000 cells/well in 96 well microtiter plates and 24 hrs laterdrugs were added and serial diluted. The cells were incubated at 37° C.for 72 hrs at which time the tetrazolium dye, XTT, containing phenazinemethosulfate was added. A dehydrogenase enzyme in live cells reduces theXTT to a form that absorbs light at 450 nm which can be quantitatedspectrophotometrically. The greater the absorbance the greater thenumber of live cells, The results are expressed as an IC50 which is thedrug concentration required to inhibit cell proliferation (i.e.absorbance at 450 nm) to 50% of that of untreated control cells.

The results for selected compounds of the present invention are shown inTable II.

TABLE II Example P388 Example P388 No. IC₅₀ (μM) No. IC₅₀ (μM) 2 0.038813 0.0232 1 0.093 14 6.22 18 0.1233 16 0.31 4 <0.003 7 0.4928 3 0.0146 80.0053 11 0.07 12 0.1066 17 0.24 6 0.0141

What is claimed is:
 1. A compound of Formula (I)

or pharmaceutically acceptable salt or solvate thereof wherein, R ishydrogen, OH, OC₁₋₇alkyl, NH₂, N(C₁₋₃alkyl)₂ or C₁₋₇alkyl, wherein saidC ₁₋₇alkyl is optionally substituted with one or more substituentsselected from the group consisting of halogen, CN, OR⁹ and NR⁹R¹⁰; Q isO, S, CH₂ or NR^(5a); one of R⁵ and R^(5a) is Formula (A):

and the other is hydrogen; R¹, R², R³ and R⁴ are each independentlyselected from the group consisting of hydrogen, C₁₋₇alkyl,C₃₋₇cycloalkyl, halogen, azido, NR⁹R¹⁰, NHC(O)NR⁹R¹⁰, NHC(O)OR⁹,C(O)OR⁹, SR⁹ and OR⁹, wherein said C₁₋₇alkyl is optionally substitutedwith one or more substituents selected from the group consisting ofhalogen, CN, OR⁹, SR⁹ and NR⁹R¹⁰; or R¹ and R² together form ═N—OH, ═Oor —NR⁹R¹⁰; or R³ and R⁴ together form ═N—OH, ═O or —NR⁹R¹⁰; W isselected from the group consisting of hydrogen, C₁₋₇alkyl,C₃₋₇cycloalkyl, halogen, azido, NR⁹R¹⁰, NHC(O)NR⁹R¹⁰, NHC(O)OR⁹, N—OH, Oand OR⁹, wherein said C₁₋₇alkyl is optionally substituted with one ormore substituents selected from the group consisting of halogen, CN, OR⁹and NR⁹R¹⁰; R⁷ and R⁸ are independently OH or H or together form ═O; R⁹and R¹⁰ are independently selected from the group consisting ofhydrogen, C₁₋₇alkyl and C₃₋₇cycloalkyl, wherein said C₁₋₇alkyl isoptionally substituted with one or more substituents selected from thegroup consisting of halogen, CN, OH, O—C₁₋₇alkyl, NH₂ and N(C₁₋₃alkyl)₂;or R⁹ and R¹⁰ together with the nitrogen atom to which they are attachedform a non-aromatic 5-8 membered heterocycle containing one or two ofthe same or different heteroatoms selected from the group consisting ofO, N and S; and X¹, X^(1′), X² and X^(2′) are independently selectedfrom the group consisting of hydrogen, halogen, cyano, OR⁹, —CF₃,alkylcarbonyl, C-₁₋₇alkyl, nitro, NR⁹R¹⁰, SR⁹ and C(O)OR⁹; wherein saidC₁₋₇alkyl is optionally substituted with one or more substituentsselected from the group consisting of halogen, CN, OR⁹, SR⁹ and NR⁹R¹⁰.2. The compound of claim 1, wherein one of R^(5a) and R⁵ is Formula (A).3. The compound of claim 2, wherein Q is NH or S.
 4. The compound ofclaim 2, wherein R is H, OH, or NH₂.
 5. The compound of claim 2, whereinR⁷ and R⁸ together are ═O.
 6. The compound of claim 2, wherein X^(2′)and X² are each F and X¹ X^(1′) are each H.
 7. The compound of claim 2,wherein X^(2′) is F and X², X¹ and X^(1′) are each H.
 8. The compound ofclaim 2, wherein X^(2′), X², X¹ and X^(1′) are each F.
 9. The compoundof claim 2, wherein X^(2′) and X² are each H and X¹ and X^(1′) are eachF.
 10. The compound of claim 2, wherein R¹, R², R³ and R⁴ are eachindependently selected from the group consisting of H, F and ORH. 11.The compound of claim 2, wherein W is F.
 12. A method of inhibitingtopoisomerase 1 to inhibit tumor growth in a mammalian host in needthereof, comprising administering to said host a tumor-growth inhibitingamount of a compound of claim 1.